Clostridium difficile is the principal cause of nosocomially acquired diarrhea in the U.S., and a number of large outbreaks of C. difficile- associated disease have been reported in the literature. To help track nosocomial acquisition and transmission in the Clinical Center, we have developed a simple, yet discriminatory technique for typing isolates of C. difficile. Conventional molecular approaches to typing microorganisms (for example plasmid analysis, restriction endonuclease analysis, and pulsed-field gel electrophoresis) are either relatively non-discriminatory, or time consuming, or both. To enable rapid assessment of the genetic relatedness of C. difficile isolates, we amplified the 16S to 235 intergenic spacer region of the ribosomal RNA gene cluster by PCR (PCR ribotyping). C. difficile possesses multiple alleles of this gene cluster, each differing in intergenic spacer length, and possession of the different alleles appears to vary in a 'strain' dependent manner. Use of this technique enabled us to assist the Hospital Epidemiology Service in identifying, retrospectively, an outbreak of C. difficile-diarrhea on 13W, and we are using the technique in a prospective manner to help identify future outbreaks.